The hypoosmotic swelling test in fresh goat spermatozoa

Transcription

1 Anim. Reprod., v.2, n.2, p , April/June, 2005 The hypoosmotic swelling test in fresh got spermtozo J.F. Fonsec 1,3, C.A.A. Torres 2, V.V. Mffili 2, A.M. Borges 2, A.D.F. Sntos 2, M. T. Rodrigues 2, R.F.M. Oliveir 2 1 Embrp Cprinos, CP D10, CEP , Sobrl, CE, Brsil 2 Deprtmento de Zootecni, Universidde Federl de Viços, Av. P. H. Rolfs, s/n, CEP , Viços, MG, Brsil. Abstrct The hypoosmotic swelling test (HOST) hs been proved to be good tool for evluting the membrne integrity of spermtozo of vrious domestic nimls including cttle, horses, nd swine. However, the best pproch for using this technique in got semen hs not been tested. The present study imed to estblish the best hypoosmotic solution (HS) for testing membrne integrity in fresh got semen. Sodium citrte nd fructose bsed solutions (S) with the following osmolrities (mosm/l) were used: 50 (S1), 75 (S2), 100 (S3), 125 (S4), 150 (S5), 175 (S6), 200 (S7), 250 (S8), 290 (S9) nd 300 mosm/l (S10). Twenty-eight semen smples were collected from seven mture bucks (four collections per buck) t 48 hour intervls. After mcroscopic evlution, 10μl of semen ws immeditely dded to 2ml of ech solution nd incubted for one hour t 37ºC. Sequentilly, 20μl of semen diluted in HS were evluted with oil immersion using phse-contrst microscope. A totl of 200 spermtozo were counted in t lest five different fields, nd sperm tils were clssified s non-coiled, coiled, nd strongly coiled. The respective percentges of spermtozo with coiled tils (coiled plus strongly coiled) in the ten solutions listed bove were 34.1, 38.8, 45.3, 51.5, 46.8, 42.8, 38.2, 29.0, 19.4 nd 23.1%. Percentges of strongly coiled spermtozo were: 6.8, 10.6, 21.5, 25.3, 24.3, 21.5, 19.3, 12.4, 6.4, nd 7.9 for the ten solutions, respectively. According to totl coiling, S4 ws superior to S1, S7, S8, S9, nd S10 (P<0.05). According to strong coiling, S4 ws superior to S1, S2, S8, S9, nd S10 (P<0.05). Results suggest tht the 125 mosm/l solution would be best for use in HOST in fresh got spermtozo. Keywords: hypoosmotic swelling test, sperm, til, got Introduction The smll ruminnt industry hs experienced gret expnsion during the lst two decdes. In tropicl nd subtropicl res of Brzil, gots hve proved to be good option for production of milk, met, nd lether. As breeds expnd, ssisted reproductive technologies re necessry to ssess nd improve the efficiency of reproduction. In mle gots, semen nlyses is bsed upon methods developed in other domestic species. Thus, it becomes necessry to test nd dpt techniques to the peculirities of got semen in the cse of the hypoosmotic swelling test (HOST) tht is used to verify sperm membrne integrity. The importnce of the membrne in mintining both biochemicl nd structurl integrity of the spermtozoon is well known (Cbrit et l., 1999). When exposed to hypoosmotic solutions, biochemiclly-ctive spermtozo increse their volume in order to estblish equilibrium between the fluid comprtment within the spermtozo nd the extrcellulr environment. Swelling cuses chnges in both cell size nd shpe tht cn be evluted using phse contrst microscope (Cbrit et l., 1999). This swelling process culmintes in promoting sphericl expnsion of the cell membrne covering the til, thus forcing the flgellum to coil inside the membrne. Coiling of the til begins t the distl end of the til nd proceeds towrd the midpiece nd hed s the osmotic pressure of the suspending medi is lowered (Jeyendrn et l., 1984). There re references for the use of HOST in cttle (Corre nd Zvos, 1994; Revell nd Mrode, 1994), swine (Vsquez et l., 1997; Zou nd Yng, 2000), horses (Neild et l., 1999), nd humns (Jeyendrn et l., 1984). The best osmolrity for hypoosmotic solution hs lredy been estblished for these species. Although Fonsec et l. (2001) reported the bility of different concentrtions of fructose-citrte solutions to cuse swelling of spermtozo from frozenthwed semen of bucks, there is no reference for use of this ssy in fresh cprine semen. The objective of this study ws to estblish the most suitble hypoosmotic solution for testing membrne integrity of fresh got semen. 3 Corresponding uthor: jeferson@cnpc.embrp.br Received: My 17, 2005 Accepted: July 8, 2005

2 Fonsec et l. The hypoosmotic swelling test in fresh got spermtozo. Loction Mterils nd Methods This study ws crried out in Mrch nd April (first hlf of locl breeding seson) t the Got Section of the Deprtment of Animl Science of the Federl University of Viços (UFV), in Viços, Zon d Mt of Mins Geris, Brzil, situted t 20º45 S ltitude nd 42º51 WG longitude. The locl verge ltitude is m bove se level with dry winter nd humid summer climtes. The verge nnul temperture is 20.9º C nd nnul rinfll is 1203 mm 3. The locl breeding seson extends from Mrch to June. solutions previously described nd incubted for one hour in wter bth t 37ºC. After incubtion, 20μl of the solutions contining semen were plced on microscope slide, covered with cover glss nd evluted using phse-contrst microscope t 1000 x mgnifiction. A totl of 200 spermtozo were counted in t lest five different fields. Spermtozo were clssified s swelled (coiled) ccording to description used by Revell nd Mrode (1994). Besides totl coiling, strong coiling ws the description given when the til becme very coiled. One Snen buck ws eliminted from the experiment due to excessive seminl pthology, minly in the sperm til which could hve confounded swelling redings due to extensive coiling. Animls Eight mture bucks, with documented good fertility (used in both rtificil insemintion nd nturl mounting progrms), three Snen nd five Alpine breeds, were used. Animls were kept in individul pens nd fed corn silge nd concentrte diet twice dy ccording to requirements. Wter nd minerl slt were vilble d libitum. Hypoosmotic solution preprtion A 300 mosm/l trisodium citrte nd fructose bsed solution ws mde ccording to the method of Revell nd Mrode (1994). As proposed by Corre nd Zvos (1994), seril dilutions were crried out in tripledistilled wter yielding solutions of 10 different osmolrities (mosm/l): 50 (S1), 75 (S2), 100 (S3), 125 (S4), 150 (S5), 175 (S6), 200 (S7), 250 (S8), 290 (S9), nd 300 (S10). Solutions were liquoted into 3 ml polyethylene tubes (2ml/tube) nd then stored t -20ºC until use. Semen collection, incubtion nd evlution Semen ws collected every 48 hours between 8:00 nd 10:00.m. by mens of n rtificil vgin nd using n estrogenized femle (1ml of estrdiol benzote t 48 hour intervl subcutneous) s sucubus. Four collections were mde for ech mle. After collection, semen smples were immeditely evluted for volume, mss movement, nd progressive motility. A volume of 10μl ws gently mixed in ech of the 2ml hypoosmotic Sttisticl nlyses Dt, presented s the men ± SD, were nlyzed by two-wy nlysis of vrince (Neild et l., 1999). Friedmn s two-wy nlysis of vrince by rnks ws pplied to evlute differences mong hypoosmotic solutions, semen smples, nd bucks (BioEstt 2.0; Ayres et l., 2000). Sttisticl significnce ws set t P < Results Spermtozo, suffering from different grdes of coiling s well s the percentges of strongly coiled nd totl coiling fter exposure to different concentrtions of hypoosmotic solutions, re presented in Fig. 1. According to these dt (Fig.2), got spermtozo ppered to suffer incresing coiling from the 50 mosm/l solution nd reched mximum vlue with the 125 mosm/l solution fter which coiling begn to decrese reching the minimum score with the 290 mosm/l solution. This tendency ws noted both in the strong nd totl coiling endpoints. According to totl coiling, S4 ws superior to S1, S7, S8, S9, nd S10 (P<0.05). When considering strong coiling, S4 ws superior to S1, S2, S8, S9, nd S10 (P<0.05). There ws no interction mong solutions, collections, nd bucks (P>0.05). Dt from S3, S4, nd S5, tht hd similr incresed percentge of coiling, were grouped to ssess differences mong collections or bucks. The percentge of totl coiling incresed significntly (P<0.05) from the first to fourth semen collection (Fig. 3) nd differed (P<0.05) mong bucks (Fig. 4). 140 Anim. Reprod., v.2, n.2, p , April/June, 2005

3 Fonsec et l. The hypoosmotic swelling test in fresh got spermtozo. A B C D E F G H I J K L M N O P Q R S T U V X W Figure 1. Got spermtozo suffering from different grdes of coiling (phse contrst microscope, 1000x mgnifiction): (A) not coiled, (B-J) coiled spermtozo nd (K-W) strongly coiled spermtozo. Note tht none spermtozo hve entrpped cytoplsmic droplet. Anim. Reprod., v.2, n.2, p , April/June,

4 Fonsec et l. The hypoosmotic swelling test in fresh got spermtozo. 80 Strongly coiled Totl coiled Coiled sperm tils (%) ,b c c,b,b Solution osmolrity (mosm/l) Figure 2. Effect of different concentrtions of hypoosmotic solutions (mosm/l) on the coiling of fresh got spermtozo (men ± SD). Mens with different superscript differed significntly mong solutions (Friedmn; P<0.05). Averge percentge of coiling ,b b First Second Third Fourth Semen collections Figure 3. Averge percentge of totl coiling in fresh got spermtozo (200 cells counted per smple) exposed to hypoosmotic sodium citrte / fructose bsed solutions (grouped 100, 125 nd 150 mosm/ml) in four subsequent semen smples. Mens with different superscript differed significntly (Friedmn; P<0.05). 142 Anim. Reprod., v.2, n.2, p , April/June, 2005

5 Fonsec et l. The hypoosmotic swelling test in fresh got spermtozo. Averge percentge of coiling b 63.4,b 52.8,b 50.0,b Buck number Figure 4: Averge percentge of totl coiling in fresh got spermtozo (200 cells counted per smple) exposed to hypoosmotic sodium citrte / fructose bsed solutions (grouped 100, 125 nd 150 mosm/ml) for Alpine (33 to 42) nd Snen (44 nd 46) bucks. Mens with different superscripts differed significntly (Friedmn; P<0.05). Discussion As shown in other domestic species of nimls (Corre et l., 1997; Neild et l., 1999), got spermtozo hd similr pttern of swelling when exposed to hypoosmotic medium. It is proposed tht under such conditions, biochemiclly-ctive sperm cells, with intct membrnes, bsorb wter nd swell incresing in volume to estblish equilibrium between the fluid comprtment within the spermtozo nd the extrcellulr medium. It culmintes into sphericl expnsion of the cell membrne covering the til, thus forcing the flgellum to coil inside the membrne. Til coiling begins t the distl end of the til nd proceeds towrds the midpiece nd hed s the osmotic pressure of the suspending medi is lowered (Jeyendrn et l., 1984). In this study, totl coiling referred to the percentge of sperm cells evluted tht suffered coiling, nd the term strong coiling referred to the percentge of sperm cells tht suffered coiling with more intensity. Considering differences in the coiling intensity, it could be speculted tht the membrne integrity cn present some degree of vrition, being more evident under some hypoosmotic solutions. It is importnt to differentite loops nd bends produced by exposing norml spermtozo to hypotonic solution (coiling) from cytoplsmic droplets in principl piece. Coiling mens tht spermtozo re vible while cytoplsmic droplets re pthologicl condition (Brth nd Oko, 1989). As proposed by Jeyendrn et l. (1984), the optiml hypoosmotic solution should exert n osmotic stress lrge enough to cuse n observble increse in volume but smll enough to prevent lyses of the sperm membrne. In the present study, this ws found between 100 to 200 mosm/l. This result ws similr to the 150 mosm/l found in cttle (Revel nd Mrode, 1994), 150 mosm/l in humns (Jeyendrn et l., 1984), but different when compred to 50 to 150 mosm/l found for swine (Vzquez et l., 1997) nd 25 to 100 mosm/l for equine spermtozo (Neild et l., 1999). This justifies the need for finding the best hypoosmotic concentrtion to be used in HOST ccording to the species being evluted. Combining dt of both strong nd totl coiling, the 125 mosm/l solution yielded the best results. Moreover, t 125 mosm/l, sperm cells ppered to undergo strongest coiling. This indictes tht this solution cused more evident swelling nd subsequent coiling nd should be chosen to be used in HOST with got fresh spermtozo. It is known tht the percentge of coiling vries ccording to bulls (Prsd et l., 1999), bucks, or seson (Kle et l., 2000) nd it is strongly correlted to mss movement, progressive motility, live sperm count, totl intct crosome, sperm concentrtion (Prsd et l., 1999), nd fertility (Jeyendrn et l., 1984). In the present study, some of these findings were confirmed in tht coiling vried ccording to bucks nd semen smples. Under the conditions of this study, HOST proved to be suitble technique for testing membrne sttus of got spermtozo. It could be vluble nd prcticl tool for ccurte ssessment of Anim. Reprod., v.2, n.2, p , April/June,

6 Fonsec et l. The hypoosmotic swelling test in fresh got spermtozo. the individul sperm cell rther thn the popultion s whole. The concentrtion of 125 mosm/l ppered to be the most dequte for use in HOST for got fresh spermtozo, nd it could id the routine nlyses of got semen. Acknowledgments Authors wnt to thnk the Ntionl Council for Scientific nd Technologicl Development (CNPq, Ministry of Science nd Technology, Brzil) nd Fundção de Ampro à Pesquis do Estdo de Mins Geris (FAPEMIG) for finncil support. References Ayres M, Ayres Jr M, Ayres DL, Sntos AS BioEstt 2.0: plicções esttístics ns áres de ciêncis biológics e médics. Belém: Editor Sociedde Civil Mmiruá, MCT CNPq. pp Brth AD, Oko RJ Abnorml morphology of bovine spermtozo. Ames: Iow Stte University Press. pp.285 Cbrit E, Alvrez R, Anel E, Herráez MP The hypoosmotic swelling test performed with Counter: method to ssy functionl integrity of sperm membrne in rinbow trout. Anim Reprod Sci, 55: Corre JR, Zvos PM The hypoosmotic swelling test: its employment s n ssy to evlute the functionl integrity of the frozen-thwed bovine sperm membrne. Theriogenology, 42: Corre JR, Heersche Jr, G, Zvos PM Sperm membrne functionl integrity nd response to frozenthwed bovine spermtozo during the hypoosmotic swelling test incubtion t vrying tempertures. Theriogenology, 47: Fonsec JF, Torres CAA, Sntos ADF, Rovy H, Borges AM, Brbos LP, Mffili VV, Frg DBM The hypoosmotic swelling test in got spermtozo. Rev Brs Reprod Anim, 25: (bstrct). Jeyendrn RS, Vn Der Ven HH, Perez-Pelez M, Crbo BG, Zneveld LJD Development of n ssy to ssess the functionl integrity of the humn sperm membrne nd its reltionship to other semen chrcteristics. J Reprod Fertil, 70: Kle MM, Mnik RS, Tomer OS In-vitro ssessment of crossbred buck fertility. Indin J Anim Sci, 70: Neild D, Chves G, Flores M, Mor N, Beconi M, Agüero A Hypoosmotic test in equine spermtozo. Theriogenology, 51: Prsd JK, Kumr S, Mohn G, Shnker U, Agrwl SK Hypo-osmotic swelling tests (HOST) nd its response in fresh nd freeze-thwed semen. Indin J Anim Sci, 69: Revell SG, Mrode RA An osmotic resistnce test for bovine semen. Anim Reprod Sci, 36: Vsquez JM, Mrtinez EA, Mrtinez P, Grci- Artig C, Roc J Hypoosmotic swelling of bor spermtozo compred to other methods for nlyzing the sperm membrne. Theriogenology, 47: Zou C-X, Yng Z-M Evlution on sperm qulity of freshly ejculted bor semen during in vitro storge under different tempertures. Theriogenology, 53: CALL FOR ABSTRACTS NINTH INTERNATIONAL SYMPOSIUM ON EQUINE REPRODUCTION Submission of bstrcts for considertion for this Symposium (ISER 9) is invited t the ddress given below. Experimentl or clinicl reserch in four res will be discussed t the Symposium: The Stllion; The Non-pregnnt Mre; Conception nd Erly Development; nd The Pregnnt Mre nd Perintology. The dedline for submission of bstrcts is 1st December The Symposium will comprise 150 short communictions divided pproximtely eqully between orl nd poster presenttions. The Proceedings of ISER 9 will be published s extended bstrcts in Specil Issue of Animl Reproduction Science nd will be vilble t the meeting. Further detils re vilble on the ISER website or from the ISER Secretrit: Mrs Jn Wde R&W Communictions, Suites 3 & 4 8 Kings Court, Willie Snith Rod, Newmrket Suffolk CB8 7SG UK Tel: +44 (0) ; Fx: +44 (0) e-mil: jn.wde@rw-communictions.co.uk 144 Anim. Reprod., v.2, n.2, p , April/June, 2005